Quantitation of gold labeling and estimation of labeling efficiency with a stereological counting method.

The amount of immunolabeling over a cell compartment of an average cell was estimated by use of an adaptation of the double disector method introduced by Gundersen. The first and last sections of a stack of ultra-thin sections formed a disector in which cell number could be estimated and related to a defined reference volume to give the cell density. Another stack section, selected at random, was immunolabeled and the number of gold particles associated with unit volume reference space (gold "density") estimated in quadrats placed systematically across the section. The ratio of gold density to cell density was used to estimate the number of gold particles lying over a chosen compartment of an average cell, N(gold)/N(cell). Such estimates required neither cell volume nor section thickness measurement and were reproducible. By combining the number of gold particles per cell with estimates of the number of protein antigens per cell, the number of gold particles associated with each antigen could be found (labeling efficiency).     

Myofibrillar ATPase histochemistry of rat skeletal muscles: a "two-dimensional" quantitative approach

In histochemical investigations of skeletal muscle, the fibers are commonly classified into three types according to their staining for myofibrillar ATPase (mATPase). In serial sections of skeletal muscles from normal Wistar rats, we compared two common staining methods for mATPase: (a) an ac-ATPase technique, with pre-incubation at pH 4.7, and (b) a fixed alk-ATPase technique, using treatment with 5% paraformaldehyde followed by pre-incubation at pH 10.4. In addition, the same fibers were stained in subsequent serial sections for succinate dehydrogenase (SDH) activity. Staining intensities were objectively evaluated by microphotometric measurements of optical density. Combining both mATPase methods in consecutive serial sections ("two-dimensional approach") led to the identification of four distinct clusters of fibers: Types I, IIA, and two subgroups of Type IIB, as separated by their staining densities for fixed alk-ATPase (IIBd dark, IIBm moderate). The mean intensity of SDH staining per fiber type, as measured in the central core of the fibers, was ranked such that IIA greater than I greater than IIBd greater than IIBm. The analyzed muscles (tibialis anterior, biceps brachii) were markedly heterogeneous with respect to the topographic distribution of different fiber types. In comparison to other muscle portions, the regions containing Type I fibers ("red" portions) showed a higher IIBd vs IIBm ratio and more intense SDH staining for either subtype of the IIB fibers. The IIBd fibers probably correspond to the Type 2X fibers of Schiaffino et al.     

Novel efficient methods for measuring mesophyll anatomical characteristics from fresh thick sections using stereology and confocal microscopy: application on acid rain-treated Norway spruce needles

Recent design-based stereological methods that can be applied to thick sections cut in an arbitrary direction are presented and their implementation for measuring mesophyll anatomical characteristics is introduced. These methods use software-randomized virtual 3D probes, such as disector and fakir test probes, in stacks of optical sections acquired using confocal microscopy. They enable unbiased estimations of the mean mesophyll cell volume, mesophyll cell number in a needle, and for the first time an internal surface area of needles or other narrow leaves directly from the fresh tissue cross-sections cut using a hand microtome. Therefore, reliable results can be obtained much faster than when using a standard microtechnical preparation. The proposed methods were tested on Norway spruce needles affected for 1 year by acid rain treatment. The effect of acid rain resulted in changes of mesophyll parameters: the ratio of intercellular spaces per mesophyll cell volume increased, while needle internal surface area, total number of mesophyll cells, and number of mesophyll cells per unit volume of a needle decreased in the treated needles.     

Axotomy-induced apoptosis in adult rat primary sensory neurons

Neuronal death following unilateral axotomy of a sensory nerve has long been inferred from neuronal counts of dorsal root ganglion neurons, using the contralateral ganglia as a control. The counting methods used usually involved the counting of neuronal nucleoli and made assumptions about them which could conceivably be flawed. Very few studies have used direct observations of dying or degenerating neurons to address questions concerning the duration of the period of neuronal death or the mechanisms involved in this process. Here we describe a morphological, morphometric and histochemical study into the nature and duration of sensory neuron death following transection and ligation of the sciatic nerve at mid-thigh level in the adult rat. We show that at least some of this neuronal loss occurs by apoptosis as defined by morphological criteria and in situ end-labelling of damaged DNA. Absolute numbers of apoptotic neurons were counted from serial paraffin sections of ganglia and estimates of neuronal numbers obtained by disector analysis at 1, 2, 3 and 6 months after axotomy. Using this approach we show that axotomy-induced apoptosis begins at around 1 week and continues up to at least 6 months after axotomy.     

Quantitative Histology Using Confocal Microscopy: Implementation of Unbiased Stereology Procedures

Direct, two-dimensional counting or measuring of cells as they appear in histological sections is subject to a number of artifacts that can lead to erroneous conclusions about changes in cellular populations. Numerous correction procedures devised to compensate for these artifacts are collectively termed model-based stereology due to their reliance on a model of cell geometry for correction formulas. These corrections are valid only to the degree that the geometric model reflects cellular morphology. In addition, there are requirements for population homogeneity that are often not met in biological material. The development of design-based stereology provides a way to directly count or measure cells in three dimensions, avoiding errors (biases) and the need for assumptions regarding cell size, shape, and orientation to be validated. On this basis, these procedures are described as unbiased stereology. The recent commercial availability of semiautomated stereology systems has substantially reduced the effort and experimenter error (bias) associated with the use of design-based stereology. The optical resolution of confocal microscopy and the ability to collect registered series of focal planes is ideally suited for the three-dimensional sampling of design-based stereology. Unfortunately, stereological procedures are not available in any confocal microscope software and it is up to the user to implement these procedures. Strategies and illustrations of approaches to implementing stereological procedures on a confocal microscope are presented. Where possible, particular design issues are discussed and solutions suggested. With user requests, future generations of confocal software may integrate collection of confocal images with the implementation of design-based stereology.     

High-performance liquid chromatography analysis of naturally occurring D-amino acids in sake

We measured all of the D- and L-amino acids in 141 bottles of sakes using HPLC. We used two precolumn derivatization methods of amino acid enantiomer detection with o-phthalaldehyde and N-acetyl-L-cysteine, as well as (+)-1-(9-fluorenyl)ethyl chloroformate/1-aminoadamantane and one postcolumn derivatization method with o-phthalaldehyde and N-acetyl-L-cysteine. We found that the sakes contained the D-amino acids forms of Ala, Asn, Asp, Arg, Glu, Gln, His, Ile, Leu, Lys, Ser, Tyr, Val, Phe, and Pro. We were not able to detect D-Met, D-Thr D-Trp in any of the sakes analyzed. The most abundant D-Ala, D-Asp, and D-Glu ranged from 66.9 to 524.3 μM corresponding to relative 34.4, 12.0, and 14.6% D-enantiomer. The basic parameters that generally determine the taste of sake such as the sake meter value (SMV; "Nihonshudo"), acidity ("Sando"), amino acid value ("Aminosando"), alcohol content by volume, and rice species of raw material show no significant relationship to the D-amino acid content of sake. The brewing water ("Shikomimizu") and brewing process had effects on the D-amino acid content of the sakes: the D-amino acid contents of the sakes brewed with deep-sea water "Kaiyoushinosousui", "Kimoto yeast starter", "Yamahaimoto", and the long aging process "Choukijukusei" are high compared with those of other sakes analyzed. Additionally, the D-amino acid content of sakes that were brewed with the adenine auxotroph of sake yeast ("Sekishoku seishu kobo", Saccharomyces cerevisiae) without pasteurization ("Hiire") increased after storage at 25 °C for three months.     

Split Nucleoli as A Source of Error in Nerve Cell Counts

The number of nerve cells in a given ganglion or nucleus is usually determined by counting the nucleoli in serial sections. The possibility that nucleoli may split and appear in more than one section is recognized as a source of error. A determination of the value of this error was made as follows; from nodose ganglia of four cats were cut serial transverse sections in which the sections varied in thickness. Thus from one ganglion, four sections were cut at 12 μ, then six at 9μ, and eight at 6μ. The process was repeated over and over until the entire ganglion was sectioned. The other ganglia were sectioned similarly. After mounting and staining, separate counts were made of the nucleoli of each given ganglion from the sections of different thicknesses. If nucleoli split according to theoretical expectations, the percentage of nucleoli split in thick sections should be less than the percentage split in thinner sections and the counts based on the sections of different thicknesses should vary accordingly. The results obtained indicate that the counts from thin sections do not differ appreciably from counts from much thicker sections, i. e., the thickness of the sections does not affect the count. It is, therefore, concluded that no correction should be made for split nucleoli if the sections are around 10 μ in thickness and none but distinct and definite nucleoli are counted.     

Reliability and validity of the physical disector method for estimating neuron number

The physical disector was proposed as an unbiased and efficient means to estimate neuron number; however, the validity and reliability of this method have been examined only infrequently. Estimates of neuron number in the dorsal root ganglia (DRG) of bullfrogs (Rana catesbeiana) were compared to nucleolar counts based on 3-dimensional reconstructions. Accuracy of disector estimates were not affected by size of the animal. Similarly, disector estimates were not systematically altered when area measurements were limited to cellular regions of the DRG versus inclusion of the entire cross-sectional area. However, the recommended protocol for applying the disector resulted in sampling errors that introduced considerable variability in repeated estimates of neuron number from a single ganglion. In addition to this lack of reliability, disector estimates were consistently lower than those obtained by means of a nucleolar counting method that was calibrated against 3-dimensional reconstructions of neuronal profiles. The systematic error of the disector method was greater when ganglia were cut parallel to the long axis of the DR than when they were cut perpendicular to this axis. Increasing the sample size beyond what was recommended increased the reliability of estimates obtained with the disector; however, the bias associated with the plane of section was not reduced. These results emphasize the need for empirical validation of methods used to estimate neuron number in the tissue to which they are to be applied. © 1996 John Wiley & Sons, Inc.     

Quantitative studies on ultrathin sections of bacteria infected with bacterio phage

A quantitative relationship has been established between the number of particles, for example bacteriophages, counted in ultrathin sections of bacteria and the total number present in the whole bacterial cells. The factor F relating particles counted per section with the total number of these particles per entire bacterium could be arrived at by two methods, which proved to give results in close agreement. The first involves knowledge of the average volume of a bacterial section in proportion to the average volume of a whole bacterium; if the mean number of appearances of the same particle on consecutive sections is also known, F may then be calculated. The thickness of sections and, therefore, their volume, as well as the average number of times a single particle is sectioned could be learned by examination of serial sections. By counting the relative number of T2 phage particles which had been intersected once or twice, and relating this proportion to the known phage dimensions, the thickness of the sections was determined to be about 400 A. The second measurement of F could be made in a particular case of late phage development where the number of particles per cell was countable or titratable directly in the bacterial lysate, this number being compared with the number seen in sections of the bacteria just before lysis. The different sources of errors are discussed. The statistical error is under 20 per cent, while the systematic errors are higher and cannot yet be indicated precisely. After a very cautious estimation of the upper limits, we can state, however, that the counts made with this method are certainly reliable to well within a factor of two.     

Mitochondria. I. Fine structure of the complex patterns in the mitochondria of Pelomyxa carolinensis Wilson (Chaos chaos L.)

Some of the mitochondria in the free-living giant ameba Pelomyxa carolinensis (Chaos chaos) exhibit unusual and strikingly complex morphological patterns. A study of serial sections of these mitochondria reveals that the patterns are formed by the organization and packing of minute villi (cristae mitochondriales). The form of the individual villus is a regular soft zigzag (or wave) with a bulbous enlargement at each point of inflection ("elbow") on the wave. The pattern of the mitochondria may become increasingly complex as a result of branching and fusing of the wavy villi. Densely packed fibrillar material is sometimes present in the stroma of the mitochondria.     

Activation of ryanodine receptors by flash photolysis of caged Ca2+.

Flash photolysis of DM-nitrophen generates an extremely large [Ca2+] transient ("Ca2+ spike") at the start of each Ca2+ "step." The Ca2+ spike greatly increases the speed of activation of the ryanodine receptor channel ("supercharging") and could be responsible for apparent channel adaptation.     

Cellular receptors for lymphotoxin: correlation of binding and cytotoxicity in sensitive and resistant target cells.

The binding of radioactively labeled lymphotoxin (LT) to both lymphotoxin-sensitive and -resistant cell clones was examined. The sensitive clone had a low- capacity, high-affinity ("specific") binding component, the curve of which closely followed the cytotoxicity curve of the lymphocyte mediator. The capacity of this binding component was calculated to be about 600 molecules of LT/cell. In addition, there was a low-affinity, high-capacity ("nonspecific") binding component. In striking contrast, the high-affinity, low-capacity ("specific") component was absent or greatly diminished from the resistant clone, whereas the low-affinity, high-capacity ("nonspecific") component was present at a similar level as in the sensitive cells.These binding characteristics closely resemble those observed by us and other investigators working with a variety of steroid hormones in steroid-sensitive and- resistant cell lines.     

Structure of peptostreptococcal protein L and identification of a repeated immunoglobulin light chain-binding domain.

The gene for protein L, an immunoglobulin (Ig) light chain-binding protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus, was cloned and sequenced. The gene translates into a protein of 719 amino acid residues. Following a signal sequence of 18 amino acids and a NH2-terminal region ("A") of 79 residues, the molecule contains five homologous "B" repeats of 72-76 amino acids each. Further, toward the COOH terminus, two additional repeats ("C") were found. These are not related to the "B" repeats, but are highly homologous to each other. After the C repeats (52 amino acids each), a hydrophilic, proline-rich putative cell wall-spanning region ("W") was found, followed at the COOH-terminal end by a hydrophobic membrane anchor ("M"). Fragments of the gene were expressed, and the corresponding peptides were analyzed for Ig-binding activity. The B repeats were found to be responsible for the interaction with Ig light chains. An Escherichia coli high level expression system was adapted for the production of large amounts of two Ig-binding protein L fragments comprising one and four B repeats, respectively.     

Mitochondria : I. Fine Structure of the Complex Patterns in the Mitochondria of Pelomyxa carolinensis Wilson (Chaos chaos L.)

Some of the mitochondria in the free-living giant ameba Pelomyxa carolinensis (Chaos chaos) exhibit unusual and strikingly complex morphological patterns. A study of serial sections of these mitochondria reveals that the patterns are formed by the organization and packing of minute villi (cristae mitochondriales). The form of the individual villus is a regular soft zigzag (or wave) with a bulbous enlargement at each point of inflection ("elbow") on the wave. The pattern of the mitochondria may become increasingly complex as a result of branching and fusing of the wavy villi. Densely packed fibrillar material is sometimes present in the stroma of the mitochondria.     

Considérations quantitatives sur des coupes ultraminces de bactéries infectées par un bactériophage

A quantitative relationship has been established between the number of particles, for example bacteriophages, counted in ultrathin sections of bacteria and the total number present in the whole bacterial cells. The factor F relating particles counted per section with the total number of these particles per entire bacterium could be arrived at by two methods, which proved to give results in close agreement. The first involves knowledge of the average volume of a bacterial section in proportion to the average volume of a whole bacterium; if the mean number of appearances of the same particle on consecutive sections is also known, F may then be calculated. The thickness of sections and, therefore, their volume, as well as the average number of times a single particle is sectioned could be learned by examination of serial sections. By counting the relative number of T2 phage particles which had been intersected once or twice, and relating this proportion to the known phage dimensions, the thickness of the sections was determined to be about 400 A. The second measurement of F could be made in a particular case of late phage development where the number of particles per cell was countable or titratable directly in the bacterial lysate, this number being compared with the number seen in sections of the bacteria just before lysis. The different sources of errors are discussed. The statistical error is under 20 per cent, while the systematic errors are higher and cannot yet be indicated precisely. After a very cautious estimation of the upper limits, we can state, however, that the counts made with this method are certainly reliable to well within a factor of two.     

Dissociable influences of auditory object vs. spatial attention on visual system oscillatory activity

Given that both auditory and visual systems have anatomically separate object identification ("what") and spatial ("where") pathways, it is of interest whether attention-driven cross-sensory modulations occur separately within these feature domains. Here, we investigated how auditory "what" vs. "where" attention tasks modulate activity in visual pathways using cortically constrained source estimates of magnetoencephalograpic (MEG) oscillatory activity. In the absence of visual stimuli or tasks, subjects were presented with a sequence of auditory-stimulus pairs and instructed to selectively attend to phonetic ("what") vs. spatial ("where") aspects of these sounds, or to listen passively. To investigate sustained modulatory effects, oscillatory power was estimated from time periods between sound-pair presentations. In comparison to attention to sound locations, phonetic auditory attention was associated with stronger alpha (7-13 Hz) power in several visual areas (primary visual cortex; lingual, fusiform, and inferior temporal gyri, lateral occipital cortex), as well as in higher-order visual/multisensory areas including lateral/medial parietal and retrosplenial cortices. Region-of-interest (ROI) analyses of dynamic changes, from which the sustained effects had been removed, suggested further power increases during Attend Phoneme vs. Location centered at the alpha range 400-600 ms after the onset of second sound of each stimulus pair. These results suggest distinct modulations of visual system oscillatory activity during auditory attention to sound object identity ("what") vs. sound location ("where"). The alpha modulations could be interpreted to reflect enhanced crossmodal inhibition of feature-specific visual pathways and adjacent audiovisual association areas during "what" vs. "where" auditory attention.     

Megakaryocytes in rabbit pulmonary blood vessels

Semiserial sections of lung capillaries of seven rabbits were examined for megakaryocytes. Triads of serial sections of each lobe were examined, and megakaryocyte counts per cm2 were determined. Median megakaryocyte count was determined for each lobe. The lobal values were used to calculate the was determined for each lobe. The lobal values were used to calculate the median cell-counts for each experimental animal. Values of 0.16-0.64 megakaryocytes per cm2 were established as "normal".     

The Contribution of Lower Oxides of Osmium to the Density of Biological Specimens in Electron Microscopy

Centrifugally stratified eggs of the sand-dollar, Dendraster excentricus, have cytoplasmic structures segregated into distinct layers. Fat droplets, yolk granules, and mitochondria are separated by "hyaline" layers of protoplasm. Aggregations of particles of 150 to 200 A diameter ("heavy bodies") are found near the mitochondrial layer and a concentration gradient of free 150 to 200 A particles corresponds to a similar gradient of basophilia in thick sections. Eggs fixed in buffered osmium-tetroxide at pH 7.4 and embedded in methacrylate were sectioned and floated on a "bleaching" solution of acidified hydrogen peroxide. "Bleached" sections showed a considerable loss in general density and especially a loss in the sharp images of cellular membranes. It was shown that such loss is not due to sublimation of structure in the electron beam. Assuming that the "bleach" acts principally to reoxidize lower oxides of osmium, it was concluded that reduced and bound lower oxides of osmium play a major role in creation of the electron micrograph image, especially in the delineation of phospholipide components of cellular membranes. Particles of 150 to 200 A diameter showed little or no loss in density, but rather a high intrinsic electron density. Refractometric data were presented to substantiate the tentative conclusions.     

Cell counting and three-dimensional reconstruction to identify a cellular wave in human spermatogenesis

OBJECTIVE: To collect quantitative and stereologic data on the main cell types represented in the seminiferous tubule in order to produce a three-dimensional representation of the morphologic events of normal cell production and maturation. STUDY DESIGN: Three-dimensional reconstruction and differential cell counting were performed on serial sections of normal testicular tissue from normal young men through image analysis and computer-based rendering. RESULTS: Peculiar periodicity in the total number of tubular cells considered along the sequence of serial sections was recognized. Also, consensual periodicity pertaining to each cell type was recognized. Adjacent high cellularity and low cellularity segments included a fairly constant mixture of cell types considered. CONCLUSION: A newly defined cellular wave, composed of regular alternations of high and low cellularity segments, was identified in the normal human seminiferous tubule.